1. Field of the Invention
The present invention relates to means for detecting and identifying microorganisms in biological samples. In particular, the present invention relates to the specific genetic detection of microorganisms based on the transformation phenomenon. The present invention also relates to methods of preparing extracts of deoxyribonucleic acid from microorganisms in biological samples which are useful in such a transformation assay.
Transformation is the heritable modification of the properties of one strain of microorganism (acceptor) by deoxyribonucleic acid (DNA) extracted from the cells of another strain of microorganism (donor). The genes carried from the donor cells to the acceptor cells are incorporated into the chromosome of the acceptor cells replacing their corresponding alleles. A variant which is distinguishable from its wild, parent strain in one or more characteristics is transformed by DNA from the parent strain to form a transformant strain in which one or more of the distinguishing characteristics of the variant has disappeared and has been replaced by the corresponding characteristic of the parent strain.
For instance, an auxotroph is a mutant which differs from its parent strain in its nutrient requirements. The auxotroph requires at least one more nutrient for growth which is not required by its parent strain. DNA from the parent strain transforms the auxotroph into a transformant strain which requires no more nutrients than required by the parent strain. Such a transformant is named a prototroph.
2. Description of the Prior Art
Since the serendipitous discovery of the transformation phenomenon in 1928, the scientific community has primarily been concerned with verifying the role of DNA as the substance which causes transformation to occur and with the use of the transformation phenomenon in studying the mechanisms of genetics. DNA is now overwhelmingly held to be the transformation-causing substance. This conclusion is based on exhaustive research efforts aimed at isolating and highly purifying each cellular component and attempting to induce transformation using the resulting highly pure homogenous extracts. Up until the publication of certain aspects of the present invention in the J. Bacteriol. 112(2):917(197), all of the prior art involving the principle of DNA-extracted transformations taught that only either virtually pure DNA or DNA obtained following involved procedures could be used to induce transformation in microorganisms. Publications relating to this teaching are J. Mol. Biol. 3:208(1961), J. gen. Microbiol. 37. 341(1964), and Hayes, W. The Genetics of Bacteria and Their Viruses, John Wiley and Sons, Inc, (New York 1964) p. 55.
At the present time, the detection and identification of microorganisms usually involve tedious compilations of data relating to the characteristics of the particular microorganism being studied. Characteristics that are generally studied include the morphology of colonies, the ability of the microorganism to ferment various carbohydrates, and many more. Even on the basis of as many as 25 tests to determine characteristics exhibited by the particular microorganism being studied, a highly skilled taxonomist still must, at times, be satisfied with making an educated guess as to the identity of the microorganism. In terms of clinical diagnosis, this conventional procedure has limited utility since in order that characteristics can be identified with a homogenous colony, pure isolates must be prepared from each test sample and numerous characterising tests must be performed, and yet the taxonomist must qualify his conclusion as to the identity of the microorganism.
While the transformation phenomenon has been known for several decades, it has not been found useful in the clinical detection of microorganisms because of the technical complexities involved in conventional transformation procedures. Standard DNA purifications are highly complicated procedures requiring on the average about three days to complete.
In the diagnosis of microbial derived disease, the accurate identification of the microorganism involved often is essential to proper and effective treatment. The conventional taxonomical methods are burdened with inherent error factors and in many cases confuse rather than clarify a patient's condition. A particular case in which specific identification is urgently needed is in the detection of Neisseria gonnorhoeae. Colonies of this virulent microorganism are easily mistaken for less harmful bacteria following the conventional taxonomical methods.
A determination based solely on genetics is the only true means of detecting and identifying microorganisms. It is a truism that microorganisms are what they are because of their genetic composition. The clinician presently lacks what would be his most useful tool for diagnosing microbial derived diseases, the rapid genetic determination of microorganisms.